Starch is the major storage carbohydrate in higher plants. The biochemical mechanisms of starch biosynthesis and starch utilization are of interest for understanding fundamental aspects of plant physiology and also for their potential utility in manipulating the starch pathway for practical purposes. Not only is starch a critical primary source of dietary carbohydrates, but it is also used extensively for various industrial purposes ranging from formation of packaging materials to ethanol production. Despite its wide availability in nature and its many industrial applications, the mechanisms by which starch is formed and degraded in plant endosperm tissue are not well understood.
Starch consists essentially of a mixture of the homopolysaccharides amylose and amylopectin [11, 24]. Amylose is a linear chain of glucosyl units joined by alpha-1,4 glycosidic bonds and normally constitutes about 25% of the total endosperm starch in maize (Zea mays). Amylopectin comprises many linear chains of glucosyl monomers joined by alpha-1,4 linkages and constitutes approximately 75% of the starch. The chains of amylopectin are joined to each other by alpha-1,6 glycosidic bonds, often referred to as branch linkages. In amylopectin, the organized positioning of branch linkages enables periodic clustering of the linear chains [9, 15]. This permits tight and efficient packaging of glucose units, and confers crystallinity to the granule. The functional properties of starch relate directly to this architectural organization of linear chains and branch linkages in amylopectin [23].
The organization of amylopectin and amylose into higher order structures that lead to granule formation renders starch resistant to degradation. However, starch granules can be completely degraded by a combination of phosphorolysis and hydrolysis when glucose supply is required [2]. As no single enzyme has been shown to completely convert starch to simple sugars, multiple enzymes most likely are involved. Starch debranching enzymes and disproportionating enzymes are potential degradative enzymes, as are the alpha-1,4 linkage-specific hydrolases of the alpha-amylase and beta-amylase classes. Genetic evidence for involvement of an alpha-amylase in starch degradation comes from the sex4 mutant of Arabidopsis, which lacks such an enzyme and accumulates abnormally high levels of starch in leaves [34]. Another enzyme that likely participates in starch degradation is phosphorylase, which inserts phosphoryl groups from inorganic pyrophosphate into the alpha-1,4 glucoside bond, releasing glucose-1-phosphate.
Classification of starch degrading enzymes is made according to their behavior: endo- versus exo-mode of attack, inversion versus retention of anomeric configuration of the substrate, preference for length of the glucosyl chain, preference for the nature of the glucosyl bond, and hydrolytic versus glucosyl-transfer activity [31]. Alpha-amylases are endo-acting hydrolytic enzymes that hydrolyze internal alpha-1,4 linkages in alpha D-glucan polymers, such as amylopectin and amylose molecules. Alpha-amylases are widely distributed in nature, and are produced by plants, animals, and microorganisms [28]. Those from different sources are known to have different substrate specificities, acting preferentially on glucan chains of different lengths. This substrate specificity is dependent on the configuration of the active site of the enzyme and results in characteristic products that are formed according to the enzyme source [28]. For example, salivary gland and pancreatic alpha-amylases immediately produce low molecular weight products such as maltose and maltotriose by “multiple attack” on the substrate [27], and barley alpha-amylase primarily produces maltose, maltohexaose, and maltoheptaose without multiple attack [19]. Because alpha-amylases can hydrolyze linkages only so close to a branch point (generated by an alpha-1,6 linkage), activity halts when this physical limitation occurs. When hydrolytic activity of the alpha-amylase reaches this limit, the resulting product is termed a “limit dextrin”. To achieve further hydrolysis of the limit dextrin, other enzymes must be employed, such as exo-cleaving beta-amylases or debranching enzymes. All of the alpha-amylase activities that have been described to date hydrolyze alpha D-glucans to maltose, maltotriose, or other small malto-oligosaccharides plus alpha-limit dextrins of various sizes [19, 28, 31].
Hydrolysis of starch with alpha-amylases from bacteria or fungi is routinely used by some starch industries as a first step in the process of the complete degradation of starch to glucose (this step is termed “saccharification”). The hydrolysis of starch to glucose is preliminary to the manufacture of conversion products such as high fructose corn syrup or fuel ethanol [18]. The goal of other starch processing industries is the incomplete hydrolysis of starch by various degradative enzymes, including alpha-amylases, to generate limit dextrins (termed “maltodextrins”) in a range of sizes that are used for a variety of industrial purposes. For example, maltodextrins are used in food and pharmaceutical manufacturing as thickening agents, cryoprotectants and binders. They can also be further processed or chemically modified for use as viscosity or hygroscopicity or dissolving agents [13]. Different limit dextrin products are typically produced by varying the combination of enzymes used for the starch digestion, or by varying the digestion conditions. An important industrial goal is the low-energy production of specific starch hydrolysates containing few by-products [18].
In plants, alpha-amylases are believed to be involved in the hydrolysis of transient starch in the leaves, which occurs during the dark cycle of the plant, and in the hydrolysis of storage starch that accumulates in seeds or tubers, which occurs during seed germination or tuber sprouting. The first complete sequence of a plant genome, that of the Arabidopsis genome, reveals that three alpha-amylase genes are present in this plant species [14, 16]. Two are genes that encode predicted polypeptides of approximately 50-60 kilodaltons (kD), and one is a gene that encodes a larger form predicted to have a molecular mass of approximately 100 kD (Genbank Accession No. NM—105651). Sequencing of the rice genome reveals the presence of one homolog of the Arabidopsis gene that encodes the large alpha amylase [12]. This rice gene is also predicted to encode a polypeptide of approximately 100 kD (Genbank Accession No. AP003408). In addition, the rice genome contains several genes that code for smaller sized (50-60 kD) alpha-amylases. All of the plant 50-60 kD alpha-amylases are similar in size to those from bacteria, yeast, and mammals that are used commercially. Activities of the 50-60 kD alpha-amylase enzymes from plants also are similar to those of bacterial, fungal, and mammalian alpha-amylase enzymes, in that they result in starch hydrolysis products consisting of maltose, maltotriose, or small oligosaccharides plus alpha limit dextrins. The activities of the 100 kD plant alpha-amylases and the nature of their starch hydrolysis products have not been characterized to date.
Alignment of the amino acid residues of all predicted alpha-amylases (both large and small) reveals they are highly similar in their C-terminal regions, which are believed to contain the catalytic domain of the protein [17]. The two 100 kD alpha-amylases from Arabidopsis and rice also have considerable amino acid sequence similarity, with 37% sequence identity in their N-terminal regions and 59% amino acid identity overall. The N-termini of the Arabidopsis and rice 100 kD alpha-amylases also have two small regions of similarity with another protein from Arabidopsis that has been termed the R1-protein, the product of the sex1 gene [26, 35]. Mutations in the sex1 gene result in excess starch accumulation, suggesting that a functional R1-protein is required for starch degradation. This suggests the larger 100 kD alpha-amylases from plants comprise a distinct isoform class of alpha-amylase enzymes. Further investigation into the role of the large alpha-amylase in starch metabolism, particularly in an agronomically important plant such as maize, is desirable.